Our current research project concerns three major lines of investigation: (1) Characterization of enzymatic reaction intermediates and products. Using low temperature and inhibitors, several bacterial luciferase-flavin intermediates have been stabilized, accumulated and isolated. Some of the intermediates have already been and others will be characterized with respect to spectral properties, catalytic activities, and reaction products. The site of substrate monooxygenation in the reaction catalyzed by salicylate hydroxylase has been determined. (2) Enzyme active site studies. The method of photoaffinity labeling has been used to delineate the active site structure of both bacterial luciferase and salicylate hydroxylase. The synthesis of labeling agents, the photolabeling of active sites, and the identification of labeled amino acid residues are at various stages of progress. (3) Spectral characterization of probe. The absorption and fluorescence properties of a fluorescence NAD ion analog have been characterized.